Small molecules capable of replicating the function of natural transcriptional activators through interactions with the transcriptional machinery are powerful tools for studying gene transcription and are emerging as a new strategy for combating disease. However, mimicking the function of natural activators with small molecules has proven challenging. Only two classes of small molecules have been designed that upregulate transcription in cells, whereas only amphiphatic isoxazolidines display potent activity at nanomolar concentrations. The KIX domain of an essential co-activator, cyclic-AMP response element-binding (CREB)-protein (CBP) was recently identified as one intercellular target for isoxazolidines capable of upregulating transcription. In contrast to many co-activators, KIX is a well-folded protein domain, biophysically characterized, and is allosterically controlled through two binding sites. KIX is therefore a prime target for studying structure and binding for the design of new functional small molecules capable of regulating transcription. This proposal uses KIX as a well-characterized, multi-functional target to develop a general platform for designing molecules that reconstitute the function of natural activators and will be accomplished through three specific aims: 1) Structural replication of natural activators 2) Binding analysis of an isoxazolidine:co-activator complex and 3) Functional replication of natural activators. Planned experiments to meet these goals will use fluorescence-based binding and 2D-NMR experiments to assess the binding affinity and binding profiles of isoxazolidine interactions, as well as cell-free and cell-based reporter assays to determine the functional role of isoxazolidines for regulating transcription. Structural information will additionally be used for designing isoxazolidines that exploit the plasticity of KIX to achieve unprecedented protein-like potentiation (enhanced binding) of a second binding site through allosteric regulation of the KIX domain. Finally, binding and structural analysis of isoxazolidine:KIX interactions will be compared with isoxazolidine functional activity in cellular assays. PUBLIC HEALTH RELEVANCE: Transcription of misregulated genes is a hallmark for a variety of different disease states. Small molecules that reconstitute the function of natural activators for regulating transcription offer an exciting strategy for studying disease and gene pathways. Results from this study will be used to develop general strategies for controlling transcription using artificial transcriptional activators.